Electromagnetic radiation spectrum covers a wide wavelength range, from gamma-ray, x-ray, UV, visible light, infra-red (near and far IR), terahertz (THz), to microwave and radio wave. The energy levels in the IR (10 THz to 400 THz) and THz (0.1-10 THz) regions coincide with molecular bond vibrational and rotational energies. Therefore, in science and technology the IR and Terahertz regions of the radiation spectrum are typically used for chemical and molecular structure identification. Each chemical and biological molecule and macromolecule (protein, enzyme) has its molecular vibrational and rotational energy signatures, unique finger prints to the species, detectable and identifiable in the IR and THz spectrum region.
IR spectroscopy is widely used for chemical analysis. IR spectroscopy typically uses a “white” light (a broad band with all wave lengths included) to illuminate the sample and detect the missing (absorbed) components in the spectrum, such implicating the presence of specific vibrational chemical bonds and, therefore the presence of the chemical species. These vibrational bonds can also be probed using a single wavelength laser to detect the spectrum of the scattered laser energy (called Raman scattering, or Raman spectroscopy). With the advancement of solid state lasers, such as quantum cascade lasers, and optical filters, Raman spectroscopy has become the favored technique in micro and nano scale chemical and structural analysis, due to the accuracy and stability of modern microelectronics and optics.
Raman spectroscopy typically involves the illumination of a sample with a laser beam (with a well-defined wave length and tight half-width) and collecting the radiation scattered by the illuminated sample for analysis. LabRAM® ARAMIS from Horiba Scientific is one of such instrument with microscope and stepping function. Once illuminated by the laser the electrons in the molecules in the sample can become excited and either absorb or lose a photon. When the excited molecule returns to its ground state it will emit a photon (light) with the energy unique to the structure of the emitting molecule. If a molecule absorbs a photon and reemits, it's called Stoke's emission, otherwise, anti-Stoke's emission. With the aid of a well-focused laser Raman spectra can be obtained from small volumes and allow the identification of species present in the volumes.
Terahertz spectra lay between IR and microwave in the electromagnetic spectrum. In technology, the Terahertz (sometime called mm wave) region is also termed “Terahertz gap”. This is because there is almost no natural occurring radiation source for terahertz. IR radiation is produced by photon excitation which is best for >10 THz while microwave radiation by electron excitation good for <0.1 THz. Due to the difference in source and detection, THz spectroscopy is typically a different method from Raman spectroscopy which uses IR. Recently, however, a detection method has been developed by Ondax, Inc. which, using a volumetric filter, can combine Raman and THz in a single system. Low energy phonons can also be detected using a BragGrate™ Notch Filter from OptiGrate Corp., for example, in volumetric holographic grating.
Biological molecules and macromolecules (protein/enzyme) are typically very large molecules, containing thousands and millions of H, C, O, N, and other atoms with a well-defined and folded structure. Such large molecules not only exhibit molecular bond vibration, they also have collective motions (rotation, shear, breath, torsion). For a C—H bond, the vibrational energy is typically between 2700-3300 (l/cm) (or 80-100 THz) depending on the type of the molecule the bond resides. For C—C bond, the bond energy range (1200-1700 l/cm), (or 40-50 THz) also depending on the bond's environment. Typically, the heavier an atom the lower the vibrational energy, with all other factors being equal. IR Raman spectroscopy covers a range of 333-5000 l/cm. (10-150 THz). Energy below 333 l/cm (0.1 to 10 THz) belong to Terahertz domain. For very large molecules such as macromolecules, additional vibrational collective modes occur. These collective motions have energy levels low in the terahertz region (<333 l/cm, or <10 THz). Increasingly, THz spectroscopy in conjunction with Raman spectroscopy are used for protein and macromolecule structural analysis.
Extending Raman into the terahertz region for macromolecule (very large proteins and enzymes) structure detection, such as protein structural vibration in membrane is a new development. Currently, there is no viable method to effectively excite and detect such motion (in-vivo in membrane). Proteins and enzymes (such as lysozyme and ribosome) are very large molecules and can have their crystal structure determined by x-ray crystallography. But there is no viable method to determine their structure while they are in-vivo (in membrane). There is no direct link between their DNA/amino sequence and their enzymatic functionality. Terahertz-Raman allows for the first time for such determination. In a normal terahertz, the wave length is such that terahertz cannot focus onto a single molecule. A normal SERS Raman can detect single molecule chemically but not its macrostructure. Terahertz-Raman is to use very high resolution Raman to obtain terahertz information (molecular structure through its acoustic signature).
Raman spectroscopy (333-5000 l/cm) can be employed to detect the presence of biological and medical specimens, including organic molecules such as proteins, glucose, and insulin most efficiently in powder and in crystalline form. Raman is also used as light scattered in solution by analyte molecules is unique to the particular molecules, which allows the determination of the molecules that are present within solvent, tissue, or blood samples. For in-situ, in-vitro, and in-vivo analysis, surface enhanced Raman spectroscopy (SERS) can be used. Surface-enhanced Raman spectroscopy (SERS) is a technique that enhances Raman scattering by molecules interacting with rough metal surfaces or nanostructures. The enhancement mechanism relates to laser stimulated surface plasmon resonance in certain metals, Au, Ag, Pt, being most common. When the incident laser frequency coincides with the surface plasmon, strong absorption and reemission of the laser energy occurs, and so too the signals from the molecules present on such surfaces. Surface plasmon resonance has been widely used in bio-sensors for biomolecule antigens detection without the use of labeling agents. Advancements in nanotechnology have allowed nanoparticles suspended in solution and mixed with the chemical to be analyzed. The presence of the nanoparticles in the vicinity of such chemicals greatly enhances Raman detectability. It is, however, difficult to introduce suspended particles in vivo. Biomolecules such as proteins have been detected using SERS substrates. Nanopatterned bulk metallic glass (BMG) such as Pt-BMG can enhance glucose detection. The enhancement of signals obtained using SERS technology may include different modes from those obtained using traditional non-enhancing techniques as the symmetries of detected molecules can be changed, depending on the polarity of the laser.
The patch clamp technique is employed in the study of ion channels in cells. The technique generally involves the use of a micropipette in contact with a patch of cell membrane, for example a neuron membrane. A glass micropipette having a tip diameter of about one to ten microns and filled with a suitable electrolyte solution encloses an area of a membrane surface (a “patch”) that contains one or more ion channels. Suction may be applied to form a high-resistance seal that electrically isolates the membrane patch for “cell-attached” recording or, in some cases, to rupture the membrane for “whole-cell” recording. The micropipette is retracted following membrane contact in some variations of the patch clamp technique, namely for “inside-out” and “outside-in” recording, in order to separate the patch from the cell membrane. The various techniques allow the recording of electrical currents from a small patch or a whole cell. Micropipettes having relatively large diameters, for example 10-25 μm, have been employed for forming gigaseals in the study of electrogenic pump and exchange transport.